We are investigating the macromolecular recognition and inhibition between serine proteases and their inhibitors. In particular, we are studying the origin of the broad inhibitory specificity of ecotin, an E. coli protease inhibitor. The goal of this study is to understand the molecular basis of ecotin's inhibitor specificity and introduce novel specificity into ecotin to target disease-related serine proteases. Based on the high resolution crystal structure of trypsin-ecotin complex, we have designed a series of ecotin variants at the two loops of ecotin and tested these mutants against different proteases. The result suggests a unique mode of inhibition of ecotin through a secondary binding site that does not occur in other serine protease inhibitors. Using phage display techniques, we have isolated several potent inhibitors against a serine protease, urokinase-type plasminogen activator, that plays an important role in cancer. This experiment validates a novel approach to design specific protease inhibitors. The molecular modeling of ecotin loop mutants is accomplished w ith MidasPlus software available in the Computer Graphics Laboratory.